Developing a pragmatic consensus procedure supporting the ICH S1B(R1) weight of evidence carcinogenicity assessment.

The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.

In silico approaches in carcinogenicity hazard assessment: case study of pregabalin, a nongenotoxic mouse carcinogen.

In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.

Cardiovascular differentiation of imatinib and bosutinib in the rat.

Imatinib and bosutinib were administered to rats for up to 6 months at clinically relevant exposures to investigate the effects on the cardiovascular system. Imatinib treatment resulted in increased volume, wall thickness and mass suggesting a hypertrophic heart in male and female rats at one and fivefold clinical exposures, respectively. Bosutinib treatment resulted in milder cardiac hypertrophy in female rats only at fivefold clinical exposures. Analysis of excised hearts and cultured myocytes demonstrated increased expression of hypertrophic genes with imatinib or analogs, but not bosutinib or c-Abl RNAi treatment. The current dataset suggests that cardiovascular liability of imatinib and bosutinib are differentiated preclinically and c-Abl independent.

Epithelial tissue hyperplasia induced by the RAF inhibitor PF-04880594 is attenuated by a clinically well-tolerated dose of the MEK inhibitor PD-0325901.

Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers.

Developmental immunotoxicity (DIT) testing of pharmaceuticals: current practices, state of the science, knowledge gaps, and recommendations.

The development and regulatory approval of immunomodulatory pharmaceuticals to treat many human diseases has increased significantly over the last two decades. As discussed by FDA and ICH guidelines, all human pharmaceuticals in development should be evaluated for potential adverse effects on the immune system. Developmental immunotoxicology (DIT) focuses on the concern that early-life (during pre-/post-natal development) exposure to agents which target the immune system may result in enhanced susceptibility to immune-related disease (e.g., infection, autoimmunity, and cancer, particularly leukemia) compared to adults, unique effects not observed in adults, or more persistent effects in comparison to those following adult exposure. This article provides a substantive review of the literature and presents detailed considerations for DIT testing strategies with a specific focus on pharmaceuticals and biopharmaceuticals. In this regard, differences between small molecule and large molecule therapeutics will be considered, along with recommendations for best practices in the assessment of DIT during drug development. In addition, gaps in the DIT knowledge base and current testing strategies are identified. Finally, a summary of an ILSI-HESI-ITC sponsored Workshop conducted in 2010, entitled 'Developmental Immunotoxicity Testing of Pharmaceuticals' will be presented. This Workshop consisted of participants from the pharmaceutical, biotechnology, academic, and regulatory sectors, where many of the issues relating to DIT outlined in this review were discussed, key points of consensus reached, and current gaps in the science identified.

Identification and elucidation of the biology of adverse events: the challenges of safety assessment and translational medicine.

There has been an explosion of technology-enabled scientific insight into the basic biology of the causes of adverse events. This has been driven, in part, by the development of the various "omics" tools (e.g., genomics, proteomics, and metabolomics) and associated bioinformatics platforms. Meanwhile, for decades, changes in preclinical testing protocols and guidelines have been limited. Preclinical safety testing currently relies heavily on the use of outdated animal models. Application of systems biology methods to evaluation of toxicities in oncology treatments can accelerate the introduction of safe, effective drugs. Systems biology adds insights regarding the causes and mechanisms of adverse effects, provides important and actionable information to help understand the risks and benefits to humans, focuses testing on methods that add value to the safety testing process, and leads to modifications of chemical entities to reduce liabilities during development. Leveraging emerging technologies, such as genomics and proteomics, may make preclinical safety testing more efficient and accurate and lead to better safety decisions. The development of a U.S. Food and Drug Administration guidance document on the use of systems biology in clinical testing would greatly benefit the development of drugs for oncology by communicating the potential application of specific methodologies, providing a framework for qualification and application of systems biology outcomes, and providing insight into the challenges and limitations of systems biology in the regulatory decision-making process.

Summary of a workshop on nonclinical and clinical immunotoxicity assessment of immunomodulatory drugs.

The number of anti-inflammatory and immunomodulatory drugs being developed in the pharmaceutical industry has increased considerably in the past decade. This increase in research and development has been paralleled by questions from both regulatory agencies and industry on how best to assess decreased host resistance to infections or adverse immunostimulation caused by immunomodulatory agents such as anti-cytokine antibodies (e.g., the tumor necrosis factor-alpha inhibitors), anti-adhesion molecule antibodies (e.g., anti-alpha-4 integrin inhibitors) and immunostimulatory molecules (e.g., anti-CD28 antibodies). Although several methods have been developed for nonclinical assessment of immunotoxicity, highly publicized adverse events have brought to light significant gaps in the application of nonclinical immunotoxicity testing in assessing potential risk in humans. Confounding this problem is inconsistent application of immunotoxicology methods for risk assessment within the scientific community, limited understanding of appropriate immunotoxicity testing strategy for immunomodulators and inconsistent testing requests by regulatory agencies. To address these concerns, The Immunotoxicology Technical Committee (ITC) of the International Life Science Institute (ILSI) Health and Environmental Sciences Institute (HESI) organized a workshop on Immunomodulators and Clinical Immunotoxicology in May 2007. The Workshop was convened to identify key gaps in nonclinical and clinical immunotoxicity testing of anti-inflammatory and immunomodulatory agents and to begin to develop consistent approaches for immunotoxicity testing and risk assessment. This paper summarizes the outcome of the HESI ITC Immunomodulators and Clinical Immunotoxicology Workshop. Topics not discussed at the Workshop were outside the scope of this report. Although more work is needed to develop consistent approaches for immunotoxicity assessment of immunomodulators, this Workshop provided the foundation for future discussion.

In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3C protease.

(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (Kobs/[I]) of 223,000 M-1s-1}. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC50) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (Cmax) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC50 required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the Cmax at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that Cmax rather than the Cmin might be the key variable predicting clinical efficacy.